DNA Amplification from Microdissected Chromosomes

We will develop kits, services, and reagents useful in the processes of generating and employingamplified DNA mini-libraries (i.e., amplified products) from microdissected human chromosomefragments. The focus of Phase I studies will be development of standard protocols for generating suchmini-libraries. Two amplification strategies are proposed based on their use of recognition sitesdistributed throughout the human genome at about 250 bp intervals (insuring representative coverageof genomic sequences in the min-library). The strategies are based on DNA sequence recognition byMbo I cleavage and by oligonucleotide hybridization to the most common 6 bp sequences in the humangenome. DNA products obtained from both strategies are PCR amplified with a single adaptoroligonucleotide. The products of the amplification reactions will be analyzed for the amount and sizeof DNA, degree of genome coverage, hybridization specificity, retrievability upon reamplification, easeof use and reproducibility. Based on amplification strategies developed in Phase I, we will ultimatelymake available to the human genom research community standardized reagents that can be used: a) forobtaining region-specific clones (i.e., genomic clones for contig assembly/map closure, and/or cDNAclones to identify novel genes), b) as starting material for isolation of region-specific STSs, and c) ashybridization/amplification targets for subchromosome localization.